The remarkable ability of Staphylococcus aureus to cause acute infections can be largely attributed to the wide array of virulence factors it produces, combined with its ability to exploit various metabolic niches within a host. In contrast, its ability to cause more persistent infections is a function of its propensity to form biofilm, a process that is enhanced by the implantation of medical devices. The current funding cycle has revealed remarkable developmental processes that occur during biofilm formation, including multiple developmental stages, stochastic gene expression, and the formation of distinct structures. Based on these preliminary results, it is hypothesized that the distinct developmental stages of S. aureus biofilm development are required for optimal fitness during infection. This hypothesis will be tested in three specific aims including to 1) define the steps involved in the transition from a planktonic to sessile lifestyle, 2) provide mechanistic insight into the bistable switch, and 3) demonstrate the in vivo role of bistability. Combined, these aims will provide critical insight into the developmental steps involved in S. aureus biofilm formation and define the relevance of these processes in vivo. A common theme of this project will be the highly synergistic nature of the experiments in the context of the other projects of this program, particularly the experiments probing the regulation of virulence gene expression (Project 3) and the in vivo relevance of this regulation (Project 4). Once completed, the results of these experiments will greatly enhance our understanding of S. aureus biofilm development and provide insight into the characteristics of these complex structures that make them so recalcitrant to clinical intervention.